The frequency of opportunistic fungal infections has increased in the last decade. Invasive mycoses have emerged as major causes of morbidity and mortality (Groll, A. H. et al. (1996) J. Infect. 33: 23-32; Minamoto, G. Y. and Rosenberg, A. S. (1997) Med. Clin. North Am. 81: 381-409; Walsh, T. J., et al. (1996) Infect. Dis. Clin. North Am. 10: 365-400). The vast majority of the invasive fungal infections are caused by Aspergillus and Candida species (Denning, D. W. (1991) J. Antimicrob. Chemother. 28: 1-16). As fungal cells have a restricted set of specific metabolic pathways and because of their eukaryotic nature, selective targeting of fungal cells has not been successfully achieved. Azoles that inhibit sterol formation and polyenes that bind to mature membrane sterols have been the mainstays of antifungal therapy for two decades or more. However, the development of fluconazole resistance among different pathogenic strains and the high toxicity of amphotericin B (Alexander, B. D. and Perfect, J. R. (1997) Drugs 54: 657-678) have prompted the search for new antifungal agents that can augment or replace therapeutic strategies for mycotic infections in the near future.
The investigation of antimicrobial peptides from a wide range of biological sources, and their synthetic derivatives, is a novel approach to new antifungal agents. Antimicrobial peptides are part of the innate immunity against microbial invasion in all organisms including human and plants and their possible mode of action has been reviewed in detail (Bechinger, B. (1999) Biochem. Biophys. Acta 1462: 157-183; Tossi, A., et al. (2000) Biopolymers 55: 4-30; Oren, Z. and Shai, Y. (1998) Biopolymers 47: 451-463). It is believed that most of these peptides bind and permeate the cell membranes. Antimicrobial peptides can be classified into two groups: (i) cell selective antimicrobial peptides that act on a narrow spectrum of microorganisms such as bacteria or fungi, and (ii) non-cell selective antimicrobial peptides that can lyse both microorganisms and normal mammalian cells.
One group of non-cell selective antimicrobial peptides includes native lipopeptides. These lipopeptides possess a broad spectrum of activities including antibacterial, antifungal, antiviral and cytolytic. Some of these lipopeptides are gene-encoded and synthesized ribosomally. However, some are synthesized non-ribosomally in bacteria, yeast, or fungi during cultivation on various carbon sources.
U.S. Pat. No. 6,183,736 describes the identification and purification of two lipopeptides from the culture medium of the bacteria Bacillus subtilis. Both lipopeptides are cyclic, acidic, and have a broad range of antifungal and antibacterial activity. It is disclosed that the two lipopeptides can be used for controlling aflatoxin-producing fungi contamination in plants. U.S. Pat. No. 6,384,013 discloses other antifungal lipopeptides, produced by culturing microorganisms. These are cyclic hexapeptides to which unique fatty acid acyl groups are attached. As a result of deacylation of the native acyl groups and reacylation with the unique acyl groups, the peptides exert enhanced antifungal and antiparasitic potency against pathogenic strains of fungi.
Recently, the present applicants incorporated several D-amino acids in the α-helical cytolytic peptides pardaxin and melittin (U.S. Pat. No. 6,172,038 and Shai, Y. and Oren, Z. (1996) J. Biol. Chem. 271: 7305-7308; Oren, Z., and Shai, Y. (1997) Biochemistry 36: 1826-1835). The resulting peptides of pardaxin and melittin having both L- and D-amino acids retained high antibacterial activity, while exhibiting reduced cytotoxic effects in mammalian cells. These peptides paved the way for the design of novel peptide antibiotics comprising both D- and L-amino acids that are selective to microorganisms.
U.S. Pat. No. 6,172,038 and WO 98/37090 disclose non-natural synthetic peptides comprising both L- and D-amino acid residues designated diastereomeric peptides with a net positive charge that is greater than +1. Some synthetic peptides consist of at least one hydrophobic amino acid and at least one positively charged amino acid, in which at least one of the amino acid residues is a D-amino acid. Several diastereomeric peptides containing from 6 to 30 amino acid residues are disclosed in U.S. Pat. No. 6,172,038 and WO 98/37090. Certain 12-mer peptides that contain lysine and a hydrophobic amino acid, in which one-third of the amino acid residues of the peptide are D-amino acid residues, were further investigated and found to be potent antimicrobial peptides having reduced hemolytic activity (Oren, Z., et al. (1997) J. Biol. Chem. 272: 14643-14649; Hong, J., et al. (1999) Biochemistry 38: 16963-16973; Avrahami, D., et al. (2001) Biochemistry 40: 12591-12603). WO 02/40529 discloses additional diastereomeric peptides having antibacterial, antifungal and anti-cancer activity.
In some studies investigators attached fatty acids to antimicrobial peptides in order to improve their properties, e.g., to increase their stability in serum and/or to reduce hemolytic activity (Efron, L., et al. (2002) J. Biol. Chem. 277: 24067-24072). In all of these studies, the investigated peptides had antimicrobial activity prior to the attachment of the fatty acids. In addition, it was shown that attachment of fatty acids to magainin, a well characterized α-helical, positively charged antimicrobial peptide, can endow it with antifungal activity (Avrahami, D., et al. (2002) Biochemistry 41: 2254-2263).
Lipopeptides capable of inducing immunological responses, particularly of cytotoxic T lymphocytes, have been disclosed in U.S. Pat. No. 5,871,746. The lipopeptides according to U.S. Pat. No. 5,871,746 comprise a peptide having between 10 to 40 amino acids and at least one antigenic determinant, particularly preferred are peptides derived from viral proteins.
U.S. Pat. No. 5,837,249 discloses methods for inducing a cytotoxic T cell response in a mammalian host against a viral infection comprising administering to the host a peptide-fatty acid conjugate, the peptide having the amino acid sequence corresponding to the amino acid sequence of a fragment of a glycoprotein or protein of virus.
U.S. Pat. No. 5,583,198 discloses compounds consisting of an amino acid or a peptide linked to a tromethamine derivative or ethanolamine derivative to which one or more fatty acids are optionally linked. The fatty acids according to U.S. Pat. No. 5,583,198 may enhance the immunogenic properties of the peptides, enhance their absorption, and provide slow-release delivery.
Nowhere in the background art is it disclosed or suggested that coupling of fatty acids to positively charged peptides that are inactive or weakly active antibacterial and/or antifungal peptides may impart to the conjugates antibacterial and/or antifungal activity as well as endow the conjugates with selective cytolytic activity against tumor cells.